WO2017164230A1

WO2017164230A1 - Phosphorylated sarm1, antibody, sarm1 phosphorylation inhibitor, prophylactic or therapeutic agent for neurodegenerative diseases, screening method, modified sarm1, and use

Info

Publication number: WO2017164230A1
Application number: PCT/JP2017/011418
Authority: WO
Inventors: 村田　等; 阪口　政清; 木下　理恵; 山本　健一
Original assignee: 国立大学法人岡山大学
Priority date: 2016-03-23
Filing date: 2017-03-22
Publication date: 2017-09-28
Also published as: JPWO2017164230A1; JP2023027235A; JP7198489B2

Abstract

The present invention provides a phosphorylated SARM1 (Sterile alpha and TIR motif-containing protein 1) in which at least one Ser residue selected from the group consisting of position 54, position 548, and position 622 of SARM1 is phosphorylated.

Description

Phosphorylated SARM1, antibody, SARM1 phosphorylation inhibitor, preventive or therapeutic agent for neurodegenerative diseases, screening method, SARM1 variant and use

The present invention relates to phosphorylated SARM1, antibody, SARM1 phosphorylation inhibitor, preventive or therapeutic agent for neurodegenerative diseases, screening method, SARM1 variant and use.

SARM1 (Sterile alpha and TIR motif-containing protein 1) is a TIR adapter protein with a mitochondrial translocation signal and consists of 724 amino acids. SARM1 is strongly expressed mainly in cells of the nervous system (Non-patent Document 1), and is reported to be a causative molecule for ischemic neuronal cell death and neuronal axonal degeneration (Non-patent Documents 2 and 3). This neuronal cell death can be remarkably suppressed by knocking out SARM1, so if there is a drug targeting SARM1, neuronal cell death is suppressed, and various neurodegeneration such as Parkinson's disease and amyotrophic lateral sclerosis (ALS) It is considered that it can be applied to the treatment of diseases (Patent Document 1). However, there are currently no drugs targeting SARM1.

Patent Documents 2 to 5 disclose that an inhibitor of CDK8 or CDK19 is effective in treating cancer.

US20120328629 WO2015 / 159937 Special table 2016-501845 Special table 2016-503408 Special table 2015-506376

The present invention aims to suppress the function of SARM1 and prevent or treat neurodegenerative diseases.

The present inventor analyzed changes and functions of SARM1 in cells and found that SARM1 acts on mitochondrial respiratory chain complex V to inhibit ATP synthesis and induce cell death. We also found that c-Jun N-terminal kinase (JNK) activates SARM1 by phosphorylating at least one serine residue selected from the 54th, 548th and 622th groups of SARM1. . By developing a method to detect phosphorylation of at least one serine residue selected from the group consisting of 54th, 548th and 622th of SARM1, it can be used to confirm the active state of SARM1 and to develop inhibitors It becomes possible to go. The present inventor further phosphorylates at least one serine residue selected from the group consisting of 54th, 548th and 622th by using a region including ARM domain, SAM domain and TIR domain in SARM1. Has developed a method for detecting urine with high sensitivity. In addition, using this detection method, an inhibitor of SARM1 was found.

The present invention provides the following phosphorylated SARM1, antibody, SARM1 phosphorylation inhibitor, preventive or therapeutic agent for neurodegenerative diseases, screening method, SARM1 variant and use.
Item 1. Phosphorylated SARM1 in which at least one Ser residue selected from the group consisting of positions 54, 548 and 622 of SARM1 (Sterile alpha and TIR motif-containing protein 1) is phosphorylated.
Item 2. Item 2. The phosphorylated SARM1 according to item 1, wherein the SARM1 is derived from a human.
Item 3. An anti-phosphorylated SARM1 antibody that specifically recognizes a phosphorylated Ser residue at positions 54, 548 or 622 of SARM1 and does not recognize non-phosphorylated Ser residues at positions 54, 548 and 622 of SARM1.
Item 4. A SARM1 phosphorylation inhibitor comprising, as an active ingredient, at least one inhibitor selected from the group consisting of cyclin-dependent kinase 8 (CDK8), cyclin-dependent kinase 19 (CDK19), and JNK.
Item 5. A prophylactic or therapeutic agent for a neurodegenerative disease comprising as an active ingredient at least one inhibitor selected from the group consisting of cyclin-dependent kinase 8 (CDK8), cyclin-dependent kinase 19 (CDK19) and JNK.
Item 6. A preventive or therapeutic agent for neurodegenerative diseases, comprising as an active ingredient a SARM1 inhibitor selected from the group consisting of an anti-SARM1 antibody, a SARM1 decoy peptide and an antagonist.
Item 7. Anti-SARM1 antibody specifically recognizes non-phosphorylated SARM1 that can suppress phosphorylation at position 54, 548 or 622 of SARM1 or phosphorylated Ser residue at positions 54, 548 or 622 of SARM1 Item 7. The preventive or therapeutic agent for neurodegenerative diseases according to Item 6, which is an anti-phosphorylated SARM1 antibody that does not recognize non-phosphorylated Ser residues at positions 54, 548 and 622 of SARM1.
Item 8. SARM1 decoy peptide is a fragment peptide or modifications thereof including Ser ^54, Ser ⁵⁴⁸ or Ser ⁶²² of SARM1, prophylactic or therapeutic agent for neurodegenerative diseases according to claim 6.
Item 9. Item 9. The neurodegenerative disease prevention according to any one of Items 5 to 8, wherein the neurodegenerative disease is selected from the group consisting of Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, neuropathy, and Alzheimer's disease. Or a therapeutic agent.
Item 10. A method for screening a prophylactic or therapeutic agent for a neurodegenerative disease, comprising a step of detecting phosphorylation of at least one Ser residue selected from the group consisting of positions 54, 548 and 622 of SARM1.
Item 11. A method for screening a SARM1 inhibitor, comprising a step of detecting phosphorylation of at least one Ser residue selected from the group consisting of positions 54, 548 and 622 of SARM1.
Item 12. A SARM1 variant in which Ser corresponding to at least one selected from the group consisting of 54th, 548th and 622th human SARM1 is substituted with Glu or Asp.
Item 13. Item 13. Use of the SARM1 variant according to Item 12 for the production of a model cell or model animal for a neurodegenerative disease.

The present invention detects the active state of SARM1 and suppresses neuronal cell death by measuring phosphorylation and cell viability of at least one serine residue selected from the group consisting of 54th, 548th and 622th of SARM1. It is a technology that can search for effective substances. SARM1 has been reported to be a causative molecule for ischemic neuronal cell death and neuronal axonal degeneration, and if an inhibitor of SARM1 is developed, various factors such as Parkinson's disease and ALS caused by neuronal cell death and axonal degeneration are reported. It can be applied to the treatment of various neurodegenerative diseases. Until now, there was no technology that could measure the activity state of SARM1 in cells, so it was not possible to develop drugs targeting SARM1. The present invention enables screening for SARM1 inhibitors using SARM1 phosphorylation as an index. In the present invention, an ARM domain (28 to 405aa), SAM domain (406 to 550aa), TIR domain (551 to 724aa) contained in SARM1, or a fragment containing the 54th, 548th or 622th Ser residue Was used for phosphorylation analysis, and it became possible to detect phosphorylation of SARM1 serine 54th, 548th or 622th with high sensitivity and ease. As a TIR domain, a core region (D594 to E670 aa) containing S622, TIR BB-loop and DD-loop structures may be used (FIG. 10B).

Furthermore, CDK19 and CDK8 that enhance phosphorylation of JNK1, JNK2, JNK3, and SARM1 that phosphorylate SARM1 were found, and these phosphorylase inhibitors are promising candidates for neuronal cell death inhibitors. In addition, analysis of the phosphorylation state of at least one serine residue selected from the group consisting of serine 54th, 548th and 622th of SARM1 using neurons derived from iPS cells derived from patients with neurodegenerative diseases Can be applied to the evaluation of disease progression.

Measurement of stress-induced cell death rate under SARM1 expression suppression conditions Cell death induction by inhibition of mitochondrial respiration of SARM1 Phosphorylation of serine 548 of SARM1 Regulation of phosphorylation and activity of SARM1 by CDK19 and CDK8 Development of SARM1 inhibitors using phosphorylation as an indicator Phosphorylation of SARM1 by JNK Screening method for SARM1 phosphorylation inhibitors using SARM1-Ser ⁵⁴⁸ phosphorylation detection Phosphorylation of SARM1-Ser ⁵⁴ , Ser ⁵⁴⁸ , Ser ⁶²² Changes in binding protein by phosphorylation of SARM1 Screening method of SARM1 binding substance using SAM domain and TIR domain

In the present specification, SARM1 is a protein consisting of 724 amino acids localized in mitochondria, and has mitochondrial localization sequence, ARM, SAM, and TIR domains (FIG. 2A).

The amino acid sequence of human SARM1 is shown in SEQ ID NO: 1. The human SARM1 has a pI of 6.1 and a molecular weight of 79388.

Although SARM1 has many phosphorylable Thr and Ser residues, the present inventors have found that Ser ⁵⁴ , Ser ⁵⁴⁸ and Ser ⁶²² , preferably Ser ⁵⁴⁸ and Ser ⁶²² are phosphorylation sites. . At least one selected from the group consisting of Ser ⁵⁴ , Ser ⁵⁴⁸ and Ser ⁶²² , preferably SARM1 is activated by phosphorylating Ser ⁵⁴⁸ and / or Ser ⁶²² , inhibiting mitochondrial respiration and inducing cell death Therefore, inhibition of mitochondrial respiration is released by inhibiting phosphorylation of at least one selected from the group consisting of Ser ⁵⁴ , Ser ⁵⁴⁸ and Ser ⁶²² , preferably Ser ⁵⁴⁸ and / or Ser ⁶²² , and neuronal cell death Can be suppressed. Therefore, at least one selected from the group consisting of Ser ⁵⁴ , Ser ⁵⁴⁸ and Ser ⁶²² , preferably a phosphorylation inhibitor of Ser ⁵⁴⁸ and / or Ser ⁶²² is useful as a preventive or therapeutic agent for neurodegenerative diseases.

Examples of neurodegenerative diseases include Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis and Alzheimer's disease, and in particular, Parkinson's disease and amyotrophic lateral sclerosis (ALS).

The present inventor confirmed that phosphorylation of SARM1 is cyclin-dependent kinase 8 (CDK8) and / or cyclin-dependent kinase 19 (CDK19), and also JNK (JNK-1, JNK-2, JNK-3, particularly JNK-3) Found out to be done. By inhibiting one or both of CDK8 and CDK19 (preferably both), JNK (JNK-1, JNK-2, JNK-3, especially JNK-3), the activation of SARM1 can be suppressed, Cell death can be suppressed, and neurodegenerative diseases can be prevented or treated. Nerve cells do not regenerate after cell death even if one or both of CDK8 and CDK19 (preferably both), and also JNK (JNK-1, JNK-2, JNK-3, especially JNK-3) are inhibited, CDK8 And / or inhibitors of CDK19, and also JNK (JNK-1, JNK-2, JNK-3, especially JNK-3) are useful to prevent or delay the prevention or progression of neurodegenerative diseases. In the case of axonal degeneration, it can be regenerated with the preventive or therapeutic agent for neurodegenerative diseases of the present invention.

The phosphorylation motif of SARM1 by JNK is shown below.
Ser ^54: REV S PGAG
Ser ⁵⁴⁸ : MLH S PLPC
Ser ⁶²² : LVL S PGAL
A neurodegenerative disease can be prevented or treated with a SARM1 inhibitor selected from the group consisting of an anti-SARM1 antibody, a SARM1 decoy peptide, and an antagonist. The decoy peptides SARM1, include fragments peptide or modifications thereof including Ser ^54, Ser ⁵⁴⁸ or Ser ⁶²² of SARM1. The modified products are those in which the N-terminal amino group is acylated (acetylated, propionylated, etc.) or alkylated (methylated, ethylated, etc.), or the C-terminal carboxyl group is esterified or amidated. Can be mentioned. Such a decoy peptide can be easily designed by those skilled in the art from the amino acid sequence of human SARM1 of SEQ ID NO: 1. SARM1 antagonists include SAM domain (molecular weight about 17000), ARM domain (molecular weight about 40000), TIR domain (molecular weight about 19000), but may be lower molecular weight antagonists. Decoy peptides are encompassed by SARM1 antagonists.

Anti-SARM1 antibody effective for prevention or treatment of neurodegenerative diseases is at least one selected from the group consisting of positions 54, 548 and 622 of SARM1, preferably of Ser residues at positions 548 and / or 622. An antibody against non-phosphorylated SARM1 capable of suppressing phosphorylation or specifically recognizes at least one phosphorylated Ser residue selected from the group consisting of positions 54, 548 and 622 of SARM1, position 54 of SARM1, And anti-phosphorylated SARM1 antibody that does not recognize the non-phosphorylated Ser residues at positions 548 and 622. Peptides that can be used to make such antibodies include
RGPREVSPGAGTEVQ (peptides with phosphorylated Ser (S) residues can be used to generate anti-phosphorylated SARM1 antibodies, and peptides without Ser (S) residues phosphorylated on Ser54 Ser ⁵⁴ Can be used to generate antibodies to suppressible non-phosphorylated SARM1),
AREMLHSPLPCTGGK (Ser (S) residues phosphorylated peptides can be used to make anti-phosphorylated SARM1 antibodies, while Ser (S) residues unphosphorylated peptides have SARM1 Ser ⁵⁴⁸ phosphorylation. Can be used to generate antibodies to suppressible non-phosphorylated SARM1),
NFVLVLSPGALDKCM (peptides with phosphorylated Ser (S) residues can be used to generate anti-phosphorylated SARM1 antibodies, and peptides without Ser (S) residues phosphorylated on Ser ⁶²² Ser ⁶²² Can be used to generate antibodies to suppressible non-phosphorylated SARM1)
Peptides including, but not limited to, Ser ⁵⁴ , Ser ⁵⁴⁸ or Ser ⁶²² can be used.

Epitope herein, an "at least one phosphorylated antibodies to non-phosphorylated SARM1 capable of suppressing selected from the group consisting of Ser ^54, Ser ⁵⁴⁸ and Ser ⁶²² of SARM1", including Ser54, Ser548 or Ser622 May recognize an epitope in the vicinity of Ser54, Ser548 or Ser622 and suppress at least one phosphorylation selected from the group consisting of Ser ⁵⁴ , Ser ⁵⁴⁸ and Ser ⁶²² by antibody binding. It may be an antibody.

Examples of inhibitors of CDK8 and / or CDK19 include those described in WO2015 / 159937, US20120071477, US20120071477, WO2013 / 116786, Special Table 2015-506376, Special Table 2016-503408, and are currently known or found in the future. Any inhibitor of CDK8 and / or CDK19 is included in the present invention as a prophylactic or therapeutic agent for neurodegenerative diseases or a phosphorylation inhibitor / activation inhibitor of SARM1. Further, ARM domain SARM1, SAM domain, a fragment containing the TIR domain or Ser ^54, Ser ⁵⁴⁸ or Ser ⁶²² is because it competitively inhibit the phosphorylation of SARM1, phosphorylation inhibitor / activator inhibitor SARM1 / Useful as a preventive or therapeutic agent for neurodegenerative diseases.

Many inhibitors of JNK (JNK-1, JNK-2, JNK-3, especially JNK-3) are known and are now known or found in the future JNK (JNK-1, JNK-2, JNK- In particular, all inhibitors of JNK-3) are included in the present invention as agents for preventing or treating neurodegenerative diseases or phosphorylation inhibitors / activation inhibitors of SARM1.

Replacing Ser at position 54, 548 or 622 of SARM1 with Asp (D) or Glu (E) can enhance the inducing effect of neuronal cell death, and position 54, 548 or 622 of SARM1 By substituting Ser at the position with an amino acid that is not phosphorylated and is not acidic, such as Ala, the activation of SARM1 can be inhibited, and the effect of SARM1 inducing neuronal cell death can be reduced or eliminated. Asp ⁵⁴ or Glu ⁵⁴ variant of SARM1, Asp ^548, or Glu ⁵⁴⁸ variants, Asp ^622, or Glu ⁶²² variants, it is possible to enhance the neuronal cell death inducing effects of SARM1, 54-position, 548-position and 622-position A variant of SARM1 obtained by modifying at least one Ser selected from the group consisting of Asp (D) or Glu (E) is useful for producing a model animal or model cell of a neurodegenerative disease. For example, a non-human mammal / cell in which the wild-type SARM1 gene is replaced (knocked in) with an Asp ⁵⁴ or Glu ⁵⁴ variant gene, an Asp ⁵⁴⁸ or Glu ⁵⁴⁸ variant gene, an Asp ⁶²² or Glu ⁶²² variant gene, or Asp ⁵⁴ or A transgenic non-human mammal / cell into which a Glu ⁵⁴ variant gene, Asp ⁵⁴⁸ or Glu ⁵⁴⁸ variant gene, Asp ⁶²² or Glu ⁶²² variant gene has been introduced is a model animal / model cell of a neurodegenerative disease Included). Two or more of positions 54, 548 and 622 may be modified.

Examples of mammals include humans, mice, rats, guinea pigs, hamsters, rabbits, goats, dogs, cats, monkeys, cows and pigs. The base sequence and amino acid sequence of SARM1 of these mammals are known, or the gene can be easily obtained from the known base sequence of SARM1 to determine the amino acid sequence.

The anti-phosphorylated SARM1 antibody of the present invention comprises at least one phosphorylated SARM1 or phosphorylated Ser ⁵⁴ and / or Ser ⁵⁴⁸ and / or Ser ⁶²² selected from the group consisting of Ser ⁵⁴ , Ser ⁵⁴⁸ and Ser ⁶²² A monoclonal antibody or a polyclonal antibody produced from a hybridoma obtained by immunizing a mouse with a monoclonal antibody, preferably a monoclonal antibody. In the following, it may be abbreviated to "Ser ⁵⁴ and / or Ser ⁵⁴⁸ and / or Ser ^622" and "Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ^622".

Several hybridomas are obtained after immunization of mammals such as mice with Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² phosphorylated SARM1 or fragments thereof containing phosphorylated Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² . Antibodies required therein will react to Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² phosphorylation SARM1, a hybridoma producing an antibody which does not react to non-phosphorylated SARM1.

The anti-non-phosphorylated SARM1 antibody of the present invention is obtained from a hybridoma obtained by immunizing mice with Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² non-phosphorylated SARM1 or a fragment thereof containing non-phosphorylated Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ^622. Monoclonal antibodies or polyclonal antibodies produced, with monoclonal antibodies being preferred. After immunizing a mammal such as a mouse with Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² non-phosphorylated SARM1 or a fragment thereof comprising non-phosphorylated Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² , several hybridomas are obtained. Antibodies required therein will react to Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² unphosphorylated SARM1, a hybridoma producing an antibody which does not react in the Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² phosphorylation SARM1.

Mammals used for immunization include mice, rats, rabbits, goats and the like, with mice being particularly preferred. In order to obtain the antibody of the present invention, a hybridoma can be prepared using Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² phosphorylated / non-phosphorylated SARM1 or a phosphorylated / non-phosphorylated Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² fragment as an immunogen. after producing, it is necessary to select a hybridoma producing an antibody which specifically reacts with Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² phosphorylation / unphosphorylated SARM1. Induction of immunity can usually be performed by administering an immunogen in an amount of 1 ng to 10 mg in 1 to 5 divided doses every 10 to 14 days. After sufficient immunization, organs capable of producing antibodies (spleen and lymph nodes) are aseptically removed from mammals and used as parent strains during cell fusion. The spleen is most preferable as an organ to be removed. Myeloma cells are used as cell fusion partners. Myeloma cells include mouse origin, rat origin, human origin, etc., but mouse origin is preferred. Examples of cell fusion include a method using polyethylene glycol, a cell electrofusion method, and the like. Selection of spleen cells or myeloma cells that have not undergone cell fusion and hybridomas can be performed, for example, by culturing in a serum medium supplemented with HAT supplements.

Selection of a hybridoma that produces an antibody that specifically binds to Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² phosphorylated / non-phosphorylated SARM1 is obtained by collecting the above-mentioned culture supernatant, Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² phosphorylated / Direct ELISA on non-phosphorylated SARM1 or its fragment containing phosphorylated / non-phosphorylated Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² and non-phosphorylated / phosphorylated SARM1 on an EIA plate is preferred. As a result of direct ELISA, Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² phosphorylated / non-phosphorylated SARM1 or phosphorylated / non-phosphorylated Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² and its fragments are strongly colored, non-phosphorylated / phosphorylated A well that does not develop color with SARM1 is selected, and the cells in that well are subjected to cloning. The hybridoma corresponding to the culture supernatant is selected as a hybridoma producing an antibody that specifically reacts with Ser ⁵⁴ / Ser ⁵⁴⁸ / Ser ⁶²² phosphorylated / unphosphorylated SARM1. Cloning is an operation for selecting and unifying antibody-producing hybridomas. There are a limiting dilution method, a fibrin gel method, a method using a cell sorter, etc., but the limiting dilution method is preferred. Thereby, the hybridoma which produces the target monoclonal antibody is acquirable. By culturing the hybridoma obtained by the above method, a monoclonal antibody can be obtained in the culture supernatant.

As a screening method for a prophylactic or therapeutic agent for neurodegenerative diseases, specifically, nerve cells are induced from iPS cells derived from healthy subjects and patients with neurodegenerative diseases, a candidate drug or a solvent control group is added, and then phosphorylated SARM1 It can be performed by evaluating SARM1 phosphorylation and neuronal cell viability by immunostaining using antibodies, Western blotting, and MTS assay. Alternatively, a candidate drug or a solvent control group is administered to a neurodegenerative disease model mouse, and then a frozen section of the brain is prepared. Evaluation of the oxidation level and neuronal cell viability can be carried out to screen for preventive or therapeutic agents for neurodegenerative diseases.

SARM1 expressing cells include SARM1 with tags such as HA tag, His tag, and FLAG tag, and ARM domain, SAM domain, TIR domain with other tags, or Ser ⁵⁴ , Ser ⁵⁴⁸ , Ser ⁶²² . More preferred is a cell in which the fragment is coexpressed. Cell culture is performed by adding a candidate substance to the culture medium of such cells, immunoprecipitation of tagged SARM1 using antibody-binding beads against the tag of SARM1, and whether the antibody of the present invention is phosphorylated. It can be detected using an antibody such as a phosphorylated serine / threonine detection antibody. In the screening method of the present invention, a substance that suppresses phosphorylation of SARM1 can be selected as a target substance.

Hereinafter, the present invention will be described in more detail based on examples.

Example 1
(1) Inhibition of neuronal cell death by suppressing SARM1 expression In order to confirm whether SARM1 is involved in inducing neuronal cell death, an expression suppression experiment using siRNA was performed. 3 × 10 ⁵ human neuroblastoma SH-SY5Y cells are seeded in a 12-well plate, mixed with 8 μl of transfection reagent Lipofectamine RNAiMAX (Thermo Fisher Scientific) in 4 μl of 10 μM siRNA (QIAGEN) against control or SARM1, and finally Transfection was performed at a concentration of 20 nM. After 48 hours, add 40 μM 6-hydroxydopamine (6-OHDA), 1 mM paraquat, 1 μM rotenone, which is used as an oxidative stress reagent and induces Parkinson's disease-like symptoms. After 24 hours, add Hoechst solution (Thermo Fisher Scientific). After staining, the cell death induction rate was measured (FIG. 1). Compared with the control group, suppression of SARM1 expression significantly suppressed cell death. This result indicates that inhibition of SARM1 expression and function may suppress stress-induced neuronal cell death.

(2) Cell death induction by inhibition of mitochondrial respiration of SARM1 Next, the mechanism of SARM1 cell death induction was examined. Since SARM1 is a protein with a mitochondrial translocation signal, we conducted an experiment focusing on the function of mitochondria. As a result, we found that SARM1 inhibits mitochondrial respiration and decreases ATP synthesis. SARM1 is a protein of 724 amino acids and has a mitochondrial translocation signal, an ARM domain, two SAM domains, and a TIR domain (FIG. 2A). Each domain functions to analyze HEK293T 3 × 10 ⁵ cells were plated in 12-well plates, SARM1 (1 ~ 724aa) of the full-length, .DELTA.N excluding the mitochondrial targeting signal (28 ~ 724aa), except TIR domain 2 μg of pDNA encoding ΔA (1 to 550 aa) and ΔA (406 to 724 aa) excluding the N-terminal including the ARM domain was mixed with 4 μl of transfection reagent FuGENE-HD (Promega) for transfection . After 8 hours, the cells were peeled off, and 3 × 10 ⁴ cells were seeded again in a 96-well plate. 48 hours after transfection, cell viability and ATP amount were measured using CellTiter 96 Aqueous One Solution Cell Proliferation Assay reagent (Promega) (FIG. 2B) and CellTiter-Glo Assay reagent (Promega) (FIG. 2C). Compared to the control, SARM1 decreased cell viability. This effect was somewhat mitigated except for the mitochondrial translocation signal (ΔN), disappeared completely at ΔT, and enhanced at ΔA. Correlating with this value, the amount of ATP in the cells also changed (FIG. 2C). These results suggest that the TIR domain is important for cell death induction and inhibition of ATP synthesis, and that the region containing the ARM domain is important for suppressing the activity of SARM1. Similarly under transfection conditions, H ₂ flux analyzer XFe96 mitochondrial respiration capacity (Fig. 2D) using (SeahorseBioscience), which is one of active oxygen species using the _{_{ROS-Glo H 2 O 2 assay}} (Promega) The amount of O ₂ (FIG. 2E) was measured. Correlating with the amount of ATP, full-length SARM1 and ΔA decreased mitochondrial respiratory capacity, especially reserve respiratory capacity, and increased ΔT. The amount of H ₂ O ₂ increased with the expression of full-length SARM1 and ΔA (FIG. 2E). These results indicate that SARM1 acts on the mitochondrial respiratory chain complex to inhibit mitochondrial respiration and induces cell death by reducing ATP synthesis and inducing reactive oxygen species generation.

(3) Detection of phosphorylation at serine 548 of SARM1 Next, we analyzed how the activity of SARM1 is regulated. In the analysis, it was found that SARM1 was phosphorylated under conditions where ATP synthesis was inhibited by forced expression of SARM1. Performed HEK293T transfected cells (8 × 10 ⁵ cells / 6 well plate) in the control or SARM1-Flag (pDNA 4 μg + FuGENE-HD 8 μl), subjected to immunoprecipitation using Flag antibody-conjugated beads after 24 hours It was. Subsequently, as a result of Western blotting using a phosphorylated serine / threonine (P-Ser / Thr) detection antibody (BD Bioscience) and Flag antibody, it was detected with the P-Ser / Thr antibody at the same position as the SARM1 band. A band appeared (Fig. 3A). In order to confirm which part of SARM1 serine and threonine are phosphorylated, SARM1 is divided into three regions (ARM: 1 to 405aa, SAM: 406 to 550aa, TIR: 551 to 724aa). Phosphorylation was detected by the same method. As shown in FIG. 3B, the SAM domain was phosphorylated in the three regions. The SAM domain (406-550aa) contains 12 serines and 9 threonines. In order to identify which of these amino acids was phosphorylated, all 21 amino acids were replaced with alanine one by one and compared with the wild type (WT) SAM domain. Of the 21 amino acids, the band detected by the P-Ser / Thr antibody disappeared only when serine 548 was substituted with alanine (FIG. 3C). From these results, it was found that serine 548 is a phosphorylation site of SARM1.

(4) Enhancement of phosphorylation of SARM1 by CDK19 and CDK8 An experiment using a mass spectrometer was conducted to identify a phosphorylase that controls phosphorylation of SARM1. In HEK293T cells (8 × 10 ⁵ cells / 6 well plate) subjected to transfection control or SAM (406 ~ 550aa) -Flag ( pDNA 4 μg + FuGENE-HD 8 μl), with a Flag antibody-conjugated beads after 24 hours Immunoprecipitation was performed. Bands co-precipitated with SAM-Flag were detected by silver staining (FIG. 4A), and binding proteins were analyzed with a mass spectrometer. As a result, Cyclin dependent kinase 19 (CDK19) was identified as a protein that binds to the SAM domain. CDK19 is one of the CDK family proteins, and it is known that CDK8 exists as a paralog with similar functions. CDK19 and CDK8 (HA-CDK19, HA-CDK8) expression vectors added with an HA tag were prepared in order to confirm the binding and phosphorylation control of the SAM domain with CDK19 and CDK8. HEK293T cells (8 × 10 ⁵ cells / 6 well plate) in the control, SAM-Flag, performed transfection of HA-CDK19 and HA-CDK8 (pDNA 4 μg + FuGENE-HD 8 μl), Flag antibody binding after 24 hours Immunoprecipitation was performed using beads. As shown in FIG. 4B, CDK19 and CDK8 bound to the SAM domain and markedly enhanced SAM domain phosphorylation. From these results, it was found that CDK19 and CDK8 enhance phosphorylation of serine 548 of SARM1. Next, the cell viability when CDK19 or CDK8 was coexpressed with SARM1 was measured by MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay reagent). Cell viability was reduced by forced expression of CDK19 and CDK8, and cell viability was further reduced by co-expression with SARM1 (FIG. 4C). These results indicate that phosphorylation of SARM1 serine # 548 by CDK19 and CDK8 is important for SARM1 activation and cell death induction.

(5) Development of SARM1 inhibitor using phosphorylation as an index From the results in FIG. 4, it was found that cell death may be suppressed by inhibiting phosphorylation of serine 548 of SARM1. Therefore, we examined whether it is possible to develop inhibitors of SARM1 using the phosphorylation state of SARM1 as an index. As shown in FIG. 3, the SAM domain (406 to 550aa) of SARM1 is strongly phosphorylated in the cell, and thus may function as an antagonist that inhibits phosphorylation of full-length SARM1 in the cell. HEK293T cells (8 × 10 ⁵ cells / 6 well plate) control, HA tag-added SARM1 (SARM1-HA) and conditions for co-expression of SARM1-HA and SAM-Flag (pDNA 4 μg + FuGENE-HD 8 μl ) And 24 hours later, SARM1-HA was immunoprecipitated using HA antibody-bound beads (SIGMA). As shown in FIG. 5A, phosphorylation of SARM1 was detected when SARM1-HA alone was used, but phosphorylation was not detected when SAM-Flag was co-expressed. Furthermore, when cell viability was measured using CellTiter 96 Aqueous One Solution Cell Proliferation Assay reagent under the same transfection conditions, the rate of cell death induced by forced expression of SARM1 was reduced by co-expression of the SAM domain ( Figure 5B). Next, in order to inhibit phosphorylation of SARM1 by CDK19 / CDK8, phosphorylation was examined using Sennexin A (TOCRIS), a CDK19 / CDK8 inhibitor. HEK293T cells (8 × 10 ⁵ cells / 6 well plate) were transfected with control or SAM-Flag (pDNA 4 μg + FuGENE-HD 8 μl), and 0-10 μM Senexin A was added 8 hours later. 24 hours after transfection, immunoprecipitation was performed using Flag antibody-conjugated beads. Senexin A inhibited phosphorylation of the SAM domain in a concentration-dependent manner (FIG. 5C). Next, the effect of Senexin A on oxidative stress-induced cell death was examined. 3 × 10 ⁴ neuroblastoma SH-SY5Y cells were seeded in a 96-well plate, and 0-20 μM Senexin A and 1 mM paraquat were added. After 48 hours, cell viability was measured using CellTiter 96 Aqueous One Solution Cell Proliferation Assay reagent. As shown in FIG. 5D, Senexin A suppressed cell death induced by oxidative stress in a concentration-dependent manner. From these results, it was found that cell death induced by SARM1 can be suppressed by developing a substance that inhibits phosphorylation of SARM1.

(6) Induction of phosphorylation of SARM1 serine # 548 by JNK As shown in FIG. 4, forced expression of CDK19 and CDK8 enhanced phosphorylation of the SAM domain. To confirm whether CDK19 and CDK8 directly induce phosphorylation of the SAM domain, an in vitro kinase assay was performed. However, when the SAM domain was reacted with CDK19 or CDK8 in the presence of ATP, phosphorylation of the SAM domain could not be confirmed. Therefore, in order to find another kinase that directly phosphorylates SARM1, we examined using a kinase inhibitor. HEK293T cells (3 × 10 ⁵ cells / 12 well plate) were transfected with SAM-Flag expression vector (pDNA 2 μg + FuGENE-HD 4 μl), and after 8 hours, various kinase inhibitors (EGFR-I, MEK- I, p38-I, JNK-I, Akt-I) were added and cultured for 16 hours, and then the cells were collected and subjected to Western blotting. In screening for kinase inhibitors, a JNK inhibitor (JNK-I: JNK inhibitor VIII (Cayman)) suppressed phosphorylation of the SAM domain (FIG. 6A). Next, in order to confirm the direct phosphorylation of the SAM domain by JNK, an in vitro kinase assay was performed by reacting a SAM domain with a GST tag and a kinase (p38α, JNK1, JNK2, JNK3) in the presence of ATP. As shown in FIG. 6B, it was confirmed that JNK (particularly JNK3) directly induces phosphorylation of serine 548 of the SAM domain.

An antibody was prepared in order to specifically detect serine 548 phosphorylation of SARM1. Mice were immunized with AREMLHSPLPCTGGK phosphorylated peptide containing serine 548 to obtain hybridomas. The reactivity of the monoclonal antibody produced from the hybridoma was confirmed by ELISA, and a clone that reacted with the phosphorylated peptide and was less reactive with the non-phosphorylated peptide was selected (FIG. 6C), and the antibody was purified. The acquired SARM1 serine # 548 phosphorylation-recognizing antibody specifically recognized the phosphorylation of full-length SARM1. When Western blotting was performed using this antibody, phosphorylation of forcedly expressed SARM1 was detected, and phosphorylation was further enhanced by co-expression of JNK1-3 (FIG. 6D). In addition, serine 548 phosphorylation of endogenous SARM1 was detectable. Various stress stimuli were added to SH-SY5Y cells and cultured for 24 hours, and the cell extract was collected. Stress stimulation that induced cell death (paraquat, 6-OHDA, rotenone treatment) increased SARM1 serine 548 phosphorylation (FIG. 6E). From these results, JNK is responsible for direct phosphorylation of SARM1 serine 548. JNK is activated in a stress environment that induces cell death, phosphorylates SARM1, and induces neuronal cell death. It was thought that it contributed.

(7) Screening Method for SARM1 Phosphorylation Inhibitor Using Detection of SARM1-Ser548 Phosphorylation As shown in FIG. 6, SARM1 was phosphorylated in a stress environment and induced cell death. Therefore, if a substance that inhibits phosphorylation of SARM1 is found, it is possible that neuronal cell death induced by SARM1 can be suppressed. Therefore, a screening method for SARM1 phosphorylation inhibitors using SARM1-Ser548 phosphorylation detection was presented. Add 1 μg of GST-SAM in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl ₂ , 1.8 mM ATP, 1 mM DTT buffer, JNK3 100 ng, JNK inhibitor, decoy peptide, antibody Etc. were added and reacted at 37 ° C. for 1 hour. JNK inhibitor is JNK inhibitor VIII (Cayman), decoy peptide is peptide with AREMLHSPLPCTGGK sequence containing serine 548, mouse control IgG (mIgG) is normal mouse IgG (Wako), SARM1 antibody is peptide containing serine 548 Antibodies (No. 1, 2, 16, 18, 30, and 33) purified from hybridomas obtained by immunizing mice were used. Thereafter, 3 x SDS sample buffer was added to stop the reaction, and the phosphorylation level of SARM1 was evaluated by Western blotting. JNK inhibitor (FIG. 7A), decoy peptide (FIG. 7B), and antibody (FIG. 7C) each had an effect of suppressing SARM1-Ser548 phosphorylation. In particular, JNK inhibitors (JNK-I) and SARM1 antibodies (Nos. 1, 2, and 16) have a strong phosphorylation-inhibiting effect, and these substances suppressed cell death induced by SARM1. HEK293T cells were transfected with SARM1 pDNA, and after 8 hours, JNK inhibitors and antibodies were added and cultured for 16 hours. The same JNK inhibitor and antibody as those used for phosphorylation analysis were used. The antibody was introduced by electroporation (Neon transfection system, Thermo Fisher Scientific). Cell viability was then assessed by MTS assay. JNK inhibitors and SARM1 antibodies have the effect of suppressing cell death (Fig. 7D, E). By combining SARM1 phosphorylation detection and cell viability assessment assays, it is possible to screen for substances that inhibit SARM1 activation. It shows that there is.

(8) Induction of phosphorylation of SARM1 serine 54, serine 548 and serine 622 by JNK JNK induces phosphorylation of SARM1 serine 548, and inhibition of JNK decreases phosphorylation of SARM1 serine 548 and cell death Induction was suppressed. To confirm the effect of phosphorylation of SARM1 serine # 548 on SARM1 activity, a SARM1 mutant expression vector (S548A) in which serine # 548 was replaced with alanine was prepared and compared with wild-type SARM1 using an MTS assay. went. As a result, it was found that the activity of S548A decreased compared to wild-type SARM1, but not so large (FIG. 7A). Therefore, considering the possibility that other serine and threonine of SARM1 may be phosphorylated by JNK, we investigated the entire sequence of SARM1, and SARM1 found three motifs that could be phosphorylated by JNK (Ser ⁵⁴ : REV S PGAG, Ser ⁵⁴⁸ : MLH S PLPC, Ser ⁶²² : LVL S PGAL). Therefore, a mutant expression vector was prepared by substituting serine 54, serine 548 and serine 622 with one or more alanines, and compared with wild-type SARM1 by MTS assay. As a result, S622A most decreased the activity of SARM1, followed by S548A and S54A, and when all three sites were mutated, the cell death-inducing activity of SARM1 was almost lost (FIG. 8A). Phosphorylation of serine 54 and serine 622 could not be detected by the method using the phosphorylated serine / threonine recognition antibody shown in Fig. 3. Detection by another method using Phos-tag (Wako) Tried. Phos-tag is a substance that binds to a phosphate group, and when mixed with an SDS-PAGE gel, the phosphorylated protein migrates more slowly than the non-phosphorylated protein, and the phosphorylated protein can be detected. This method was used to compare SARM1 mutants. As shown in the upper photograph in FIG. 8B, in the detection using a normal SDS-PAGE gel, one SARM1 band is detected. However, as shown in the lower photo in Fig. 8B, in the SDS-PAGE gel containing Phos-tag, a band indicated by a fast black arrow and a band indicated by a slow white arrow appear and SARM1 is phosphorylated. Was confirmed (white arrow). Furthermore, not only serine 548 but also mutants in which serine 54 and serine 622 were substituted with alanine had a band pattern change compared to wild-type SARM1, suggesting that these serines are also phosphorylated. It was. However, since phosphorylation bands are also detected in mutants in which serine 54, 548, and 622 are all substituted with alanine, in addition to these serines, there may be amino acids that undergo phosphorylation in SARM1. It is suggested. To further clarify that serines 54, 548, and 622 are phosphorylated by JNK, SARM1 was expressed for each domain, co-expressed with JNK1-3, and Phos-tag was used. Detection was performed. As shown in Fig. 8C, it was confirmed that phosphorylation bands increased by co-expression of JNK (especially JNK3) in ARM domain containing serine 54, SAM domain containing serine 548, and TIR domain containing serine 622 It was done. From these results, JNK is thought to induce phosphorylation of serine 54, serine 548 and serine 622 of SARM1.

(9) Change of binding protein by phosphorylation of SARM1 In order to clarify the significance of phosphorylation of SARM1, the binding proteins of SARM1 mutants were compared. HEK293T cells (8 × 10 ⁵ cells / 6 well plate) were transfected with control, wild type SARM1-Flag or mutant SARM1-Flag (pDNA 4 μg + FuGENE-HD 8 μl). After 24 hours, prepare a cell extract with cell lysate containing 1% Triton-X100, perform immunoprecipitation with 20 μl of Flag antibody-bound beads (SIGMA) for 2 hours, and then add 0.1 M glycine solution (pH 2.5). The immunoprecipitate was collected. 1 M Tris solution (pH 9) and SDS solution were mixed, and then developed by SDS-PAGE. A band co-precipitated with SARM1-Flag was detected with a silver staining kit (Wako) (FIG. 9A), and the binding protein was analyzed with a mass spectrometer. As a result, full-length SARM1, including wild type and point mutations, was confirmed to bind to subunits ATP5B, ATP5C1, and ATP5O of the mitochondrial respiratory complex complex V, and the binding was reduced by the S548A mutation. On the other hand, a band with increased binding to SARM1 was detected in the vicinity of 70 kDa in ΔTIR having no SARM1 activity and in the S622A mutant with almost no activity, and this band was Heat shock protein 70 (HSP70). This binding was also confirmed by Western blotting after immunoprecipitation (FIG. 9B). These results suggest that SARM1 inhibits ATP synthesis by interacting with complex V (FIG. 2C), and that HSP70 suppresses the action. Furthermore, phosphorylation of serine 622 is important for dissociation from HSP70, and it is considered that phosphorylation of serine 548 contributes to the binding to complex V.

Assuming that SARM1 binds to complex V and inhibits ATP synthesis leads to cell death induction, excessive introduction of complex V subunits and functioning as decoys will reduce cell death induced by SARM1. It is assumed that it can be suppressed. HEK293T cells (3 × 10 ⁵ cells / 12 well plate) were co-expressed with SARM1-Flag and control, ATP5B, ATP5O pDNA (pDNA 4 μg + FuGENE-HD 8 μl), and 24 hours later, cell viability by MTS assay Was analyzed. Co-expression of ATP5B and ATP5O with SARM1 significantly suppressed SARM1-induced cell death (Fig. 9C). From this result, SARM1 binding to complex V and inhibiting ATP synthesis induced SARM1-induced cell death It is considered that it is possible to suppress the function of SARM1 by finding a substance that inhibits phosphorylation of SARM1 important for binding.

(10) Screening method for SARM1-binding substances using SAM domain and TIR domain Based on the results shown in Fig. 9, compounds that suppress phosphorylation of serine 548, which is important for binding to complex V, and serine required for dissociation from HSP70 A compound that suppresses phosphorylation of No. 622 is assumed to be an effective compound for inhibiting SARM1 activation and suppressing neuronal cell death. This has led to the development of a method for screening these substances.

A glycerol stock prepared by cloning the SAM domain into an expression vector pET28a having a T7 promoter (pET28a-SAM-Flag (6His-T7-SAM-Flag)) and transforming it into E. coli BL21 (DE3) -RP was prepared as LB-Kan. (Kanamycin 30 μg / ml) was cultured with shaking at 37 ° C. for 10 hours, and then IPTG was added to a final concentration of 1 μm and shaken at 25 ° C. for 16 hours to express the SAM domain. By sonication in 20% by mass of sucrose buffer, Escherichia coli crushed material was obtained. This crushed material is centrifuged, and the supernatant is added to a cobalt column. Then, PBS-T (Phsophate Buffered Saline with Tween 20) + 10 mM imidazole is used as the washing solution, and PBS + 500 mM imidazole is used as the eluent. As a result, the SAM domain was purified (FIG. 10A).

A partial region (D594 to E670 aa) of the TIR domain was cloned into an expression vector pGEX-6P-1 having a tac promoter (pGEX-6P-1-TIR (GST-TIR)) and transformed into E. coli BL21 (DE3) -RP. The glycerol stock prepared by conversion is cultured with shaking in LB-Amp (ampicillin 100 μg / ml) medium at 37 ° C. for 10 hours, and then IPTG is added to a final concentration of 1 μmM, and then at 25 ° C. for 16 hours. The TIR domain was expressed by shaking culture. By sonication in 20% by mass of sucrose buffer, Escherichia coli crushed material was obtained. This crushed material is centrifuged, and the supernatant is added to a Sephadex-4B column (GE Healthcare). PBS-T- (Phsophate Buffered Saline with Tween 20) is used as a washing solution, and 50 mM Tris-HCl (pH 8) + 10 mM The TIR domain was purified by purifying reduced glutathione as an eluent and further dialyzing against PBS (FIG. 10B).

Biacore sensor chip was prepared for molecular interaction analysis using the purified SAM domain. SAM domain 0.4 mg / ml dissolved in PBS was diluted 4-fold (0.1 mg / ml) with 10 mM mM acetate buffer (pH 4.5) and immobilized on a CM5 sensor chip by the amine coupling method. As a method for measuring the interaction with the analyte, HBS-EPHbuffer (GE Healthcare) was used as the running buffer, and the analyte was diluted to 20 μg / ml with the running buffer. In order to confirm the molecular interaction with the SAM domain, SARM1 antibody and SAM domain were added and analyzed (FIG. 10C). Since SAM domains dimerize, binding of SAM domains was confirmed by adding SAM domains. Antibodies recognizing the vicinity of serine 548 of several SAM domains have been found to bind stronger than SAM domains, and these antibodies can inhibit SAM domain dimerization and inhibit SARM1 function. It was suggested. On the other hand, binding to the SAM domain was not confirmed with antibodies recognizing other regions of control IgG and SARM1. From these results, it was shown that compounds that bind to the domain of SARM1 can be selected by using molecular interaction analysis by Biacore.

Currently, there are no drugs that can prevent or treat the progression of neurodegenerative diseases (Parkinson's disease, ALS, etc.).

SARM1 is one of the molecules that cause neuronal cell death and neuroaxonal degeneration observed in neurodegenerative diseases.

If the function of SARM1 can be inhibited, it may be possible to prevent and treat various neurodegenerative diseases.

However, since the activation mechanism of SARM1 in cells was unknown, there was no SARM1 inhibitor currently available.

The present inventor has found that SARM1 serine 54, serine 548 and serine 622 are phosphorylated by JNK for the activation of SARM1.

The present inventor has developed a screening method for drugs that inhibit SARM1 activation by combining the detection of phosphorylation of SARM1 and the measurement of cell viability.

The use of the present invention makes it possible to develop SARM1 inhibitors that are promising for the prevention and treatment of neurodegenerative diseases.

Claims

Phosphorylated SARM1 in which at least one Ser residue selected from the group consisting of positions 54, 548 and 622 of SARM1 (Sterile alpha and TIR motif-containing protein 1) is phosphorylated.
The phosphorylated SARM1 of claim 1, wherein the SARM1 is derived from a human.
An anti-phosphorylated SARM1 antibody that specifically recognizes a phosphorylated Ser residue at positions 54, 548 or 622 of SARM1 and does not recognize non-phosphorylated Ser residues at positions 54, 548 and 622 of SARM1.
A SARM1 phosphorylation inhibitor comprising, as an active ingredient, at least one inhibitor selected from the group consisting of cyclin-dependent kinase 8 (CDK8), cyclin-dependent kinase 19 (CDK19), and JNK.
A prophylactic or therapeutic agent for a neurodegenerative disease comprising as an active ingredient at least one inhibitor selected from the group consisting of cyclin-dependent kinase 8 (CDK8), cyclin-dependent kinase 19 (CDK19) and JNK.
A preventive or therapeutic agent for neurodegenerative diseases, comprising as an active ingredient a SARM1 inhibitor selected from the group consisting of an anti-SARM1 antibody, a SARM1 decoy peptide and an antagonist.
Anti-SARM1 antibody specifically recognizes non-phosphorylated SARM1 that can suppress phosphorylation at position 54, 548 or 622 of SARM1 or phosphorylated Ser residue at positions 54, 548 or 622 of SARM1 The preventive or therapeutic agent for neurodegenerative diseases according to claim 6, which is an anti-phosphorylated SARM1 antibody that does not recognize unphosphorylated Ser residues at positions 54, 548 and 622 of SARM1.
SARM1 decoy peptide is a fragment peptide or modifications thereof including Ser 54, Ser 548 or Ser 622 of SARM1, prophylactic or therapeutic agent for neurodegenerative diseases according to claim 6.
The neurodegenerative disease according to any one of claims 5 to 8, wherein the neurodegenerative disease is selected from the group consisting of Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, neuropathy and Alzheimer's disease. Prophylactic or therapeutic drug.
A method for screening a prophylactic or therapeutic agent for a neurodegenerative disease, comprising a step of detecting phosphorylation of at least one Ser residue selected from the group consisting of positions 54, 548 and 622 of SARM1.
A method for screening a SARM1 inhibitor, comprising a step of detecting phosphorylation of at least one Ser residue selected from the group consisting of positions 54, 548 and 622 of SARM1.
A SARM1 variant in which Ser corresponding to at least one selected from the group consisting of 54th, 548th and 622th human SARM1 is substituted with Glu or Asp.
Use of the SARM1 variant according to claim 12 for the production of a model cell or model animal of a neurodegenerative disease.